Single-Molecule Protein and Peptide Sequencing

ABSTRACT

The present description provides methods, assays and reagents useful for sequencing proteins. Sequencing proteins in a broad sense involves observing the plausible identity and order of amino acids, which is useful for sequencing single polypeptide molecules or multiple molecules of a single polypeptide. In one aspect, the methods are useful for sequencing multiple polypeptides. The methods and reagents described herein can be useful for high resolution interrogation of the proteome and enabling ultrasensitive diagnostics critical for early detection of diseases.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/789,850, filed on Jan. 8, 2019. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND

Proteins serve critical structural and dynamic functional roles at thecellular level of all living organisms. Understanding proteincontribution to biological function is critical and rests on havingappropriate technologies for quantification and identification. Thecentral dogma of molecular biology, information flow from DNA to RNA toprotein, has been studied for decades as these molecules are critical tocell function and diversity. The advent of polymerase chain reaction(PCR) amplification of nucleic acid was pivotal in advancing thehigh-throughput molecular interrogation and analysis of DNA and RNA atthe whole-genome and transcriptome level. In contrast, studying proteinshas lagged technologically since there is no equivalent of PCR toamplify and detect low-copy number proteins. Instead, protein sequencingand identification methods have relied on ensemble measurements frommany cells which masks cell-to-cell variations. While some researchershave turned to transcriptomics as a proxy to the protein compositionwithin cells, it is critical to note that gene expression at thetranscriptomic level weakly correlates with the proteomic profile due tovariability in translational efficiency of different mRNAs, and thedifference between mRNA and protein lifetimes. In addition,post-translational modifications also result in significant variabilityof protein abundance and their primary sequence with respect to thetranscriptome. Vital biological processes such as synaptic plasticity,metabolic signaling pathways and stem cell differentiation, all dependon protein expression. Many diseases also originate from geneticmutations that are in turn translated to a single or set of aberrantproteins. Diseases such as cancer and neurodegeneration tend to havetriggered mutations of unclear origins and polygenic interactions. Theycan be best understood and addressed at the proteomic level, since theirpathology is directly related to disrupted proteostasis at the cellularlevel.

Advancements in proteomics have lagged behind while DNA sequencing hasrapidly advanced the study of genomics primarily due to technologiesthat allow for high-throughput sequencing. Current methodologies forstudying proteins include Mass Spectrometry, Edman sequencing andImmunohistochemistry (IHC).

Mass spectrometry enables protein identification and quantificationbased on the mass/charge ratio of peptide fragments, which can bebioinformatically mapped back to a genomic database. While thistechnique has made significant advancements, it has yet to quantify acomplete set of proteins from a biological system. The technologyexhibits attomole detection sensitivities for whole proteins andsubattomole sensitivities after fractionation. The sensitivity of massspectrometry is limiting since low copy-number proteins that make upabout 10% of mammalian protein expression remain undetected and arefunctionally important despite low abundance.

The other method used for protein sequencing is the Edman degradationreaction. Edman degradation allows for sequential and selective removalof single N-terminal amino acids, subsequently identified via HPLC,High-Performance Liquid Chromatography. Edman protein sequencing is aproven method to selectively remove the first N-terminal amino acid foridentification in which phenyl isothiocyanate (PITC) is used toconjugate to the N-terminal amino acid, then upon acid and heattreatment, the PITC-labeled N-terminal amino acid is removed. AlthoughEdman sequencing can have 98% efficiency, a major drawback is that it isinherently low throughput, requiring a single highly purified proteinand not applicable to systems-wide biology. Both Edman degradation andmass spectrometry can sequence proteins but lack single moleculesensitivity and do not provide spatial information of proteins in thecontext of cells.

In regards to spatial information, immunohistochemistry is a proteinidentification method that allows us to visualize cellular localizationof proteins but does not provide sequence information.Immunohistochemistry involves the identification of proteins viarecognition with fluorophore-conjugated antibodies. This approachexcludes protein sequence information but can identify proteins andtheir respective localizations. A major limitation is the scalability,since even the perfect construction of specific antibodies for everyprotein in the proteome would require around 25,000 antibodies and,˜6250 rounds of four-color imaging. Any 1-to-1 protein tagging schemewill likely fail to scale to the entire proteome.

A major obstacle in protein sequencing is the lack of natural enzymesand biomolecules that probe amino acids on a peptide. For example, theredoes not exist protein amplification processes analogous to PCR fornucleic acid, so the approach to sequencing via single-moleculestrategies is appropriate, requiring the detection of individual aminoacids.

Current proposed approaches to single molecule protein sequencing relyeither on fluorescent read-out via covalent chemical modifications ofpeptide or protein residues, probing with N-terminal-specific amino-acidbinders (NAABs), or translocating peptides through a nanopore with avoltage applied across the membrane. Chemical modifications of aminoacids on the internal peptide chain may be vulnerable to lowefficiencies due to steric hindrance caused by adjacent chemical labels,and there is also a limited number of available reactive amino acids andchemistries for labeling of all 20 amino acids. A major issue usingnanopores for protein sequencing can be attributed to the non-uniformcharge distribution of amino acid residues and the analytical challengeof deconvolving electric recordings to discriminate between amino acids.

In the case of N-terminal amino-acid binders, peptides are immobilizedto substrates by the C-terminus so the N-terminus is accessible tobinders and sequential Edman degradation. Engineering highly specific,strong N-terminal binders that are not affected by the presence ofvariable neighboring amino acids found across different peptides ischallenging. Neighboring amino acids may affect N-terminal binding byintroducing variation in charge, sterics and secondary structure. Thiscan be referred to as the “local environment” problem. For example, whenattempting to recognize the N-terminal amino acid of a peptide, thecombinations of varying amino acids on the rest of the peptide resultsin many possible sequences that impose inconsistent interactions with anN-terminal binder.

The lack of technology for high-resolution protein-level analysesrepresents a significant gap in advancing important biological research.

SUMMARY OF THE INVENTION

The invention provides a method for identifying the terminal amino acidof a peptide. In embodiments, the method comprises contacting thepeptide with a ClickP compound, wherein the ClickP compound binds to aterminal amino acid or a terminal amino acid derivative of the peptideto form a ClickP-peptide complex, tethering the ClickP-peptide complexto a substrate; cleaving the complex from the peptide thereby providinga ClickP-amino acid complex bound to the substrate; and detecting theClickP-amino acid complex.

The invention also provides a method for identifying the terminal aminoacid of two or more peptides in a sample. In embodiments, the methodcomprises independently affixing the two or more peptides to anattachment point on a substrate; contacting the peptides with ClickPcompounds, wherein the ClickP compounds bind to a terminal amino acid orterminal amino acid derivative to form a ClickP-pepetide complex,tethering the ClickP-peptide complexes to the substrate; cleaving theClickP-peptide complexes from the peptide thereby providing aClickP-amino acid complex bound to the substrate; and detecting theClickP-amino acid complexes.

The invention also provides a method for sequencing of at least aportion of a peptide. In embodiments, the method comprises contactingthe peptide with a ClickP compound, wherein the ClickP compound binds toa terminal amino acid or terminal amino acid derivative of the peptideto form a ClickP-peptide complex, tethering the ClickP-peptide complexto a substrate; cleaving the ClickP-peptide complex from the peptide toform a ClickP-amino acid complex; detecting the ClickP-amino acidcomplex; identifying the amino acid of the ClickP-amino acid complex;releasing the ClickP-amino acid complex from the substrate; andrepeating these steps.

The invention also provides a method for sequencing at least a portionof two or more peptides in a sample independently affixed attachmentpoints on a substrate. In embodiments, the method comprises contactingthe two or more peptides with a ClickP compounds, wherein the ClickPcompounds bind to a terminal amino acid or terminal amino acidderivative to form a ClickP-peptide complexes, tethering theClickP-peptide complexes to the substrate; cleaving the ClickP-peptidecomplexes from the peptide to form ClickP-amino acid complexes;detecting the ClickP-amino acid complexes; identifying the amino acid ofthe ClickP-amino acid complexes; releasing the ClickP-amino acidcomplexes from the substrate; and repeating these steps.

The invention also provides a ClickP-amino acid complex. In embodiments,the ClickP-amino acid complex comprises a ClickP compound bound to oneof 20 natural proteinogenic amino acids; a ClickP compound bound to apost-translationally modified amino acid; or a ClickP compound bound toa derivative of (a) or (b).

The invention also provides a ClickP-amino acid complex binder. Inembodiments, the ClickP-amino acid complex binder comprises a binderthat binds to a subgroup of the 20 natural proteinogenic amino acidscomplexed with ClickP; a binder that binds to a subgroup ofpost-translationally modified amino acids complexed with ClickP; or abinder that binds to a derivative of (a) or (b).

In embodiments, the ClickP-amino acid complex binder comprises a binderthat binds to one of 20 natural proteinogenic amino acids complexed withClickP; a binder that binds to a post-translationally modified aminoacids complexed with ClickP; or a binder that binds to a derivative of(a) or (b).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 depicts one example of a ClickP compound of Formula I comprisingPITC as the primary amine reactive and cleavage group and an alkyne asthe tethering group, an azide-thiol linker, a thiol-functionalizedsurface.

FIG. 2 depicts a workflow for single molecule peptide sequencing andN-terminal amino acid identification using ClickP.

FIG. 3A through FIG. 3C depict the efficiency of ClickP candidates toconjugate and cleave the N-terminal primary amine when compared to PITC.FIG. 3A depicts N-terminal conjugation efficiency. FIG. 3B depictsN-terminal conjugation efficiency time course. FIG. 3C depictsN-terminal cleavage efficiency.

FIG. 4 demonstrates the activity of the tethering group of the ClickPcompound.

FIG. 5A and FIG. 5B depict examples of a ClickP compound bound to all 20natural amino acids.

FIG. 6A and FIG. 6B depict the local environment problem of a tryptophantargeting antibody and its ability to selectively targetClickP-tryptophan over other ClickP-amino acid complexes.

FIG. 7 depicts the mass spectrometry results of ClickP conjugation andcleavage of N-terminal amino acid.

DETAILED DESCRIPTION

The present description provides methods, assays and reagents useful forsequencing proteins. Sequencing proteins in a broad sense involvesobserving the plausible identity and order of amino acids.

In one aspect, the methods are useful for sequencing single polypeptidemolecules or multiple molecules of a single polypeptide. In one aspect,the methods are useful for sequencing multiple polypeptides.

In one aspect, the methods and reagents are useful for determining theN-terminal amino acid of a polypeptide. In one aspect, the methods areuseful for the simultaneous sequencing of a plurality of singlepolypeptide molecules, such as for the basis of massively parallelsequencing techniques. Accordingly, samples comprising a mixture ofdifferent proteins can be assayed according to the methods describedherein to generate sequence information regarding individual proteinmolecules in the sample. In a further aspect, the methods are useful forprotein expression profiling in complex samples. For example, themethods are useful for generating both quantitative (frequency) andqualitative (sequence) data for proteins contained in a sample.

In one embodiment, the invention allows for single-moleculeidentification and sequencing of proteins. The methods and reagentsdescribed herein can be useful for high resolution interrogation of theproteome and enabling ultrasensitive diagnostics critical for earlydetection of diseases.

In one aspect, the invention provides compounds, compositions, andmethods for identifying the terminal amino acid of a peptide. In oneembodiment, the invention provides reagents for N-terminal amino acidisolation and identification, such as an N-terminal amino acid isolationreagent and N-terminal amino acid isolation reagent-amino acid complexbinders. In one embodiment, the invention provides reagents forC-terminal amino acid isolation and identification, such as a C-terminalamino acid isolation reagent and C-terminal amino acid isolationreagent-amino acid complex binders. In one embodiment, a N-terminalamino acid is identified. In one embodiment, a C-terminal amino acid isidentified.

The N-terminal or C-terminal amino acid isolation reagents are alsoreferred to herein as “ClickP”. In one embodiment, the ClickP compoundhas the structure of Formula I:

wherein

A is a terminal amino acid reactive and cleaving group;

B is a releasable group;

C is a tetherable group; and

L1 and L2 are independent spacers.

The terminal amino acid reactive group reacts to and binds the terminalamino acid of a peptide. When used for N-terminal amino acid isolationthe terminal amino acid reactive group of the ClickP compound comprisesa primary amine reactive group that conjugates to the free amine at theN-terminal end of the peptide to form a ClickP-peptide complex.

When used for C-terminal amino acid isolation the terminal amino acidreactive group of the ClickP compound comprises a C-terminal reactivegroup that conjugates to the modified or unmodified carboxylic group atthe C-terminal end of the peptide to form a ClickP-peptide complex.

In embodiments, the terminal amino acid reactive group is a primaryamine reactive group. In one embodiment, the primary amine reactivegroup includes, but not limited to, isothiocyanate, phenylisothiocyanate (PITC), isocyanates, acyl azides, N-hydroxysuccinimideesters (NHS esters), sulfonyl chlorides, aldehydes, glyoxals, epoxides,oxiranes, carbonates, aryl halides, imidoesters, carbodiimides,anhydrides, and fluorophenyl esters. In one embodiment, the reagent isphenyl isothiocyanate (PITC).

In certain embodiments, the N-terminal amino acid, or derivativethereof, and the ClickP compound can be contacted under conditions thatallow the N-terminal amino acid to conjugate to the primary aminereactive group of the ClickP to form a complex.

In one embodiment, the terminal amino acid reactive group is aC-terminal reactive group. In one embodiment, the C-terminal reactivegroup includes, but is not limited to, isothiocyanate,tetrabutylammonium isothiocyanate, diphenylphosphoryl isothiocyanate,acetyl chloride, cyanogen bromide, isothiocyanate, sodium thiocyanate,ammonium thiocyanate, and carboxypeptidases.

In certain embodiments, the C-terminal amino acid, or derivativethereof, and the ClickP compound can be contacted under conditions thatallow the modified or unmodified C-terminal amino acid to conjugate toC-terminal reactive group of the ClickP, to form a complex.

In some embodiments, the cleaving group is the same as the terminalamino acid reactive group. In one embodiment, the N-terminal cleavinggroup is involved in the chemical removal of the terminal amino acidfrom the peptide. In one embodiment, the N-terminal cleaving group isinvolved in the chemical removal of the terminal amino acid from thepeptide to forms the ClickP-amino acid complex. In one embodiment, thecleaving group is PITC or isothiocyanate. In one embodiment, thecleaving group is assisted by engineered or wildtype enzymes such aspeptidases or proteases.

In one embodiment, the ClickP amino acid complex is the ClickP compoundconjugated to the amino acid following cleavage from the peptide. In oneembodiment, the ClickP amino acid complex can be chemically derivatizedto be antigenic. In one embodiment the ClickP-amino acid complex can be,but is not limited to, the following derivatized forms: thiazolone,thiohydantoin, or thiocarbamyl.

In some embodiments, the functions of reacting to amines and cleavingthe terminal amino acid from the peptide can be performed by the primaryamine reactive group. In some embodiments, the primary amine reactivegroup having both of these functions includes, but is not limited to,isothiocyanate, phenyl isothiocyanate (PITC). In one embodiment, theprimary amine reactive group is phenyl isothiocyanate (PITC). In oneembodiment, the primary amine reactive group is isothiocyanate.

In some embodiments, the functions of reacting to the C-terminus andcleaving amino acids can be performed by the same chemical group. In oneembodiment, the C-terminal cleaving group is involved in the chemicalremoval of the terminal amino acid from the peptide to forms theClickP-amino acid complex. In one embodiment, the cleaving group isisothiocyanate, tetrabutylammonium isothiocyanate, or diphenylphosphorylisothiocyanate.

In one embodiment, the tethering group includes, but is not limited to,isothiocyanate, tetrabutylammonium isothiocyanate, diphenylphosphorylisothiocyanate, azide, alkyne, Dibenzocyclooctyne (DB CO), maleimide,succinimide, thiol-thiol disulfide bonds, Tetrazine, TCO, Vinyl,methylcyclopropene, a primary amine, a carboxylic acid an alkyne,acryloyl, allyl, and an aldehyde.

The tethering group can conjugate to a functionalized substrate such asa functionalized glass surface or integrated into a polymer networkunder conditions that allows for conjugation, thereby immobilizing theClickP-peptide complex on the substrate.

In embodiments, the releasable group is involved in the removal of partor all of the ClickP-amino acid complex. In some embodiments, theClickP-amino acid complex can be released from the substrate undercertain substrate release conditions which are not the bindingconditions or the amino acid release conditions. In some embodiements,the releasable group can be but is not limited to a disulfide, peptide,oligonucleotide, or carbohydrate.

The term “substrate release conditions” refers to release conditions inwhich a ClickP-amino acid complex will be released from a substrate. Thesubstrate release conditions can include, but are not limited to, acidicconditions, basic conditions, presence of a nucleophile, presence of aLewis base, presence of a non-nucleophilic base, presence of anucleophilic base, presence of a thiol, oxidation conditions, reductionconditions, presence of a catalyst, presence of an engineered orwildtype enzyme, exposure to visible light, exposure to ultravioletlight, or combinations thereof. Release conditions can include, but notlimited to, aqueous solvents (such as water), organic solvents (such asdioaxane, DMSO, THF, DMF, Toluene, acetonitrile), or combinationsthereof. In certain embodiments, acidic conditions can include the useof hydrofluoric acid (HF), or hydrochloric acid (HCl). In certainembodiments, basic conditions can include the use of pyridine, ammonia,piperidine, 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine(DIEA), piperizine, morpholine, dicyclohexylamine, triethylamine, ordiethylamine.

By way of explanation, and not intended to limit the invention, the“cleaving” group of the ClickP compound acts to remove the terminalamino acid from the peptide while the “releasable” group provides amechanism to release the ClickP-amino acid complex from the substrate.Removing the ClickP-amino acid complex from the substrate allows for theidentification of sequential amino acids.

In some embodiments, a spacer is used to provide sufficient stericseparation between the functional groups of the ClickP compound to avoidinterference with reaction kinetics. In some embodiments the spacerincludes, but is not limited to, polymers and biopolymers such aspolyethylene glycol (PEG) chains, Aminohexanoic acid (Ahx),12-amino-dodecanoic acid, O2Oc, O1Pen-O1Pen, Ttds, Beta-Alanine,hydrocarbon chains, amino acids, peptides, peptide bonds, and nucleicacid.

In one embodiment, the ClickP compound can comprise a releasable groupbefore the tethering group.

In one embodiment, the ClickP compound can comprise a releasable groupafter the tethering group.

In one embodiment, the ClickP compound can comprise a releasable groupthat is reversible with the ability to both tether and be cleaveddependent on the condition.

-   -   Example of a releasable group with tethering group include but        are not limited to:        -   spacer-alkyne-azide-spacer-thiol-thiol-sub state,        -   spacer-thiol-thiol-spacer-alkyne-azide-sub state; and        -   spacer-thiol-thiol-substrate;

wherein the underlined portion is the functionalized substrate tetheringthe ClickP tethering group. The thiol-thiol group is a releasable groupthat releases the part of or the whole ClickP complex under certainconditions from the substrate. The releasable group can be before orafter the tethering group. In the case of thiol, it can act as both thetethering group and the releasable group.

The ClickP compound comprises a reactive group that conjugates to theterminal amino acid of the peptide; a tethering group that immobilizesthe ClickP-peptide complex to a physical substrate; and a cleaving groupthat allows for the removal of the ClickP compound and the boundterminal amino acid from the peptide resulting in a ClickP-amino acidcomplex; and a releasable group that allows for the release of thecomplex from the physical substrate.

In one embodiment ClickP compound conjugates to the terminal amino acidof the peptide to form the ClickP-peptide complex. The ClickP-peptidecomplex is then locally tethered to a physical substrate. TheClickP-peptide complex is subsequently cleaved from the peptideresulting in a ClickP-amino acid complex. After detection and/oridentification of the amino acid of the ClickP-amino acid complex, theClickP-amino acid complex can optionally be released from the substrateto allow for following consecutive rounds of sequencing. In someembodiments, the tethering group is that same as the releasable group.

In some embodiments, the ClickP-amino acid complex is antigenic. In someembodiments, a portion of the ClickP-amino acid complex is antigenic.The antigenic portion will include the attached amino acid and thefollowing portions from Formula I—only A, A and B, A and C, or A B andC. In embodiments, the antigenic portion will include the attached aminoacid and A from Formula I. In embodiments, the antigenic portion willinclude the attached amino acid and A and B from Formula I. Inembodiments, the antigenic portion will include the attached amino acidand A and C from Formula I. In embodiments, the antigenic portion willinclude the attached amino acid and A, B, and C from Formula I.

In one embodiment, Formula II depicts a portion of ClickP such that thereleasable functional group can be attached later to provide flexibilityto test various releasable linkers.

wherein n is is any number from 0 to 500. In one embodiment, n is anynumber from 0 to 250. In one embodiment, n is any number from 0 to 100.In one embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50. In one embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In oneembodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment,n is 1, 2, 3, 4, or 5. In one embodiment, n is 1.

In one embodiment, the ClickP compound can tether directly to afunctionalized surface of a substrate. For example, if the functionalizesurface is an azide containing surface, then a ClickP compoundcomprising a group that conjugates to azides, e.g., alkynes, can tetherdirectly to the surface. The conditional copper-catalyzed (Cu+) clickchemistry of alkyne-azide bonds is bioorthgonal with a high yield andhigh reaction specificity suitable for isolating target molecule incomplex biological environments.

The contacting and binding of components in a ClickP complex, or aClickP complex-substrate complex can occur in a solvent including, butnot limited to, aqueous solvents (such as water) or organic solvents(such as dioaxane, DMSO, THF, DMF, Toluene, acetonitrile).

FIG. 1 shows one example of a ClickP compound of Formula I comprisingPITC as the terminal amine reactive and cleaving group and an alkyne asthe tethering group, an azide-thiol linker, and a thiol-functionalizedsurface. As shown in FIG. 1, PITC can bind to the terminal amino acid ofa peptide to form a ClickP-peptide complex. The alkyne group conjugatesto the azide tetherable group of the azide-thiol linker, which forms adisulfide bond with a thiol-functionalized surface. The alkyne group onClickP allows for the addition of any modular azide linker such as, butnot limited to, an azide-thiol, to form bonds to various types offunctionalized surfaces. The disulfide bond allows ClickP to be releasedfrom surfaces with a reducing agent such astris(2-carboxyethyl)phosphine (TCEP) that cleaves disulfide bonds. Areleasable group for the removal of ClickP-bound amino acid from thesurface allows for the isolation and identification of the next,terminal, amino acid on a peptide.

In one embodiment, the invention provides a method for isolating aminoacids using compounds to tether the terminal amino acid of the peptideto a physical substrate and to cleave the terminal amino acid to then beidentified free from the peptide. The isolation of the terminal aminoacid from the peptide allows for more selective and/or higher affinitybinding of amino acids that is not influenced by the rest of thepeptide.

In one embodiment, identifying the terminal amino acid of a peptidecomprises contacting the peptide with a ClickP compound, wherein theClickP compound binds to a terminal amino acid or a terminal amino acidderivative of the peptide to form a ClickP-peptide complex TheClickP-peptide complex is tethered to a substrate. After tethering, theClickP-peptide complex is cleaved from the peptide to form theClickP-amino acid complex. The ClickP-amino acid complex can then beused for the detection and/or identification of the amino acid of theClickP-amino acid complex.

In one embodiment, the invention provides a method for the isolation andidentification of N-terminal amino acids, or derivatives thereof, of apolypeptide or protein. Isolation of N-terminal amino acids with ClickPwill involve conjugation of the ClickP compound to the N-terminal aminoacid of a polypeptide or a derivative thereof, to form a ClickP-peptidecomplex; conditional tethering of the ClickP-peptide complex to asubstrate, cleavage of the ClickP-peptide complex from the peptideforming the ClickP-amino acid complex; and detection and/oridentification of the ClickP-amino acid complex.

In one embodiment, the invention provides a method for the isolation andidentification of C-terminal amino acids, or derivatives thereof, of apeptide. Isolation of C-terminal amino acids with ClickP will involveconjugation of the ClickP compound to the C-terminal amino acid of apeptide or a derivative thereof, to form a ClickP-peptide complex;conditional tethering of the ClickP-peptide complex to a substrate,cleavage of the ClickP-peptide complex from the peptide, to form aClickP-amino acid complex; and detection and/or identification of theClickP-amino acid complex.

In one embodiment, there is provided a method for identifying theterminal amino acid of a plurality of peptides in a sample.

In one embodiment, the method comprises affixing the plurality ofpeptides in the sample to a plurality of attachment points on afunctionalized substrate; contacting the peptides with a plurality ofClickP compounds, wherein the ClickP compounds bind to a terminal aminoacid or N-terminal amino acid derivative to form a ClickP-peptidecomplexes, tethering the ClickP-peptide complexes to the substrate;cleaving the ClickP-peptide complexes from the peptide to formClickP-amino acid complexes; and detecting and/or identifying the aminoacids of the ClickP-amino acid complexes.

In embodiments of the methods disclosed herein, the methods optionallycomprise washing away excess and/or unbound ClickP compound prior to thestep of cleaving the ClickP-peptide complex from the polypeptide orprotein.

Sequencing of peptides with ClickP will involve conjugation of theClickP compound to the terminal amino acid of a peptide or a derivativeof a terminal amino acid of a peptide to form a ClickP-peptide complex;conditional tethering of the ClickP-peptide complex to a substrate,cleavage of the ClickP-peptide from the peptide to form a ClickP-aminoacid complex; detection and identification of the ClickP-amino acidcomplex; and release of the immobilized ClickP-amino acid complex fromthe substrate for the next cycle.

In one embodiment, detecting and/or identifying the amino acid of theClickP-amino acid complex comprises contacting the ClickP-amino acidcomplex with a ClickP-amino acid complex binder, wherein theClickP-amino acid complex binder binds to a ClickP-amino acid complex ora subgroup of ClickP-amino acid complexes; and detecting theClickP-amino acid complex binder bound to the ClickP-amino acid complex.Detecting binding of the binder to the ClickP-amino acid complex allowsfor the identification of the terminal amino acid of the peptide.

In one embodiment, detecting and/or identifying the amino acid of theClickP-amino acid complex comprises contacting the ClickP-amino acidcomplex with a plurality of ClickP-amino acid complex binders, whereineach ClickP-amino acid complex binder preferentially binds to a specificClickP-amino acid complex or a subgroup of ClickP-amino acid complexes;and detecting the ClickP-amino acid complex binder bound to theClickP-amino acid complex. By detecting the ClickP-amino acid complexbinder bound to the ClickP-amino acid complex allows for identifying theterminal amino acid or subgroup of amino acids of the peptide.

It has been determined that ClickP and ClickP-amino acid complex binderscan be used to generate sequence information by identifying the terminalamino acids of a peptide. The inventors have also determined that byfirst affixing the peptide molecule to a substrate, it is possible todetermine the sequence of that immobilized peptide by iterativelydetecting the ClickP-amino acid complex at that same location on thesubstrate.

In one embodiment, detecting and/or identifying the amino acid of theClickP-amino acid complex can comprise direct detection throughwavelengths of light. In one embodiment, ramam spectrum from singleClickP-amino acid complexes are detected to identify the complex. In oneembodiment, surface enhanced Raman spectroscopy is used to detect and/oridentify the ClickP-amino acid complex. In one embodiment, the Ramanspectrum for each ClickP-amino acid complex is distinguishable from oneanother. In one embodiment, the Raman spectrum for each ClickP-aminoacid complex are partially distinguishable from one another. In someembodiments, gold or silver can be deposited onto the substrate as aform of surface enhancement for Raman spectroscopy. In one embodiment,surface enhancement for Raman spectroscopy are nanoparticles thatinteract with ClickP-amino acid complexes. In one embodiment, theinteraction of the nanoparticles to ClickP-amino acid complexes are, butnot limited to, covalent, hydrophilic or hydrophobic interaction.

As used herein, the terms “peptide”, “polypeptide” or “protein” are usedinterchangeably herein and refer to two or more amino acids linkedtogether by a peptide bond. The terms “peptide”, “polypeptide” or“protein” includes peptides that are synthetic in origin or naturallyoccurring. As used herein “at least a portion of the peptide” refers to2 or more amino acids of the peptide. Optionally, a portion of thepeptide includes at least: 5, 10, 20, 30 or 50 amino acids, eitherconsecutive or with gaps, of the complete amino acid sequence of thepeptide, or the full amino acid sequence of the peptide.

The phrase “N-terminal amino acid” refers to an amino acid that has afree amine group and is only linked to one other amino acid by a peptidebond in the peptide. The phrase “N-terminal amino acid derivative”refers to a N-terminal amino acid residue that has been chemicallymodified, for example by an Edman reagent or other chemical in vitro orinside a cell via a natural post-translational modification (e.g.phosphorylation) mechanism, or a synthetic amino acid.

The phrase “C-terminal amino acid” refers to an amino acid that has afree carboxylic group and is only linked to one other amino acid by apeptide bond in the peptide. The phrase “C-terminal amino acidderivative” refers to a C-terminal amino acid residue that has beenchemically modified, for example by a chemical reagent in vitro orinside a cell via a natural post-translational modification (e.g.phosphorylation) mechanism, or a synthetic amino acid.

The phrase “subgroup of ClickP-amino acid complexes” refers to a set ofamino acids that are bound by the same ClickP-amino acid complex binder.In the broadest sense, the identity of the amino acid or subgroup isencoded in the binder. If the binder is not specific to one amino itmay, for example, bind to 2 or 3 amino acids with some statisticalregularity. This type of information is still relevant for proteinidentification since narrowing down the possibility of an amino acid isstill relevant for database searches. Amino acid identity and bindingvariation is based on features like polarity, structure, functionalgroups and charge which can influence the specificity of the binder.Overall, the groups are based on the binder specificity and what theyrepresent. A binder could bind two or more amino acids equally or with avarying degree of confidence, still providing sequence information.

As used herein, the binding of a binder to the ClickP-amino acid complexor subgroup of ClickP-amino acid complexes, refers to any covalent ornon-covalent interaction between the binder and the ClickP-amino acidcomplex. In one embodiment, the binding is covalent. In one embodiment,the binding is non-covalent.

As used herein, “sequencing a peptide” refers to determining the aminoacid sequence of a peptide. The term also refers to determining thesequence of a segment of a peptide or determining partial sequenceinformation for a peptide. Partial sequencing of a peptide is stillpowerful and sufficient to discriminate protein identity when mappedback to available databases. For example, it is possible to uniquelyidentify 90% of the human proteome by sequencing six (6) consecutiveterminal amino acids of a protein. In instances where a ClickP-aminoacid complex binder that binds to a subgroup of ClickP-amino acidcomplexes, the binders may not provide exact identity of the terminalamino acid but instead the plausible subgroup identity. Plausiblesequence identity information is still powerful and sufficient todiscriminate protein identity when mapped back to available databases.

As used herein, “affixed” refer to a connection between a peptide and asubstrate such that at least a portion of the peptide and the substrateare held in physical proximity. The terms “affixed” or “tethered”encompass both an indirect or direct connection and may be reversible orirreversible, for example the connection is optionally a covalent bondor a non-covalent bond.

In one embodiment, the substrate is a flat planar surface. In anotherembodiment, the substrate is 3-dimensional and exhibits surfacefeatures. In one embodiment the surface is a functionalized surface. Insome embodiments, the substrate is a chemically derivatized glass slideor silica wafer.

As used herein “the cleaving the N-terminal amino acid or N-terminalamino acid derivative of the peptide” refers to a chemical reactionwhereby the N-terminal amino acid or N-terminal amino acid derivative isremoved from the peptide while the remainder of the peptide remainsaffixed to the substrate.

As used herein “the cleaving the C-terminal amino acid or C-terminalamino acid derivative of the peptide” refers to a chemical reactionwhereby the C-terminal amino acid or C-terminal amino acid derivative isremoved from the peptide while the remainder of the peptide remainsaffixed to the substrate.

As used herein the term “sample” includes any material that contains oneor more polypeptides. Samples may be biological samples, such asbiopsies, blood, plasma, organs, organelles, cell extracts, secretions,urine or mucous, tissue extracts and other biological samples of fluidsboth natural or synthetic in origin. The term sample also includessingle cells. The sample may be derived from a cell, tissue, organism orindividual that has been exposed to an analyte (such as a drug), orsubject to an environmental condition, genetic perturbation, orcombination thereof. The organisms or individuals may include, but arenot limited to, mammals such as humans or small animals (rats and micefor example).

In one embodiment, the attachment points on the functionalized surfaceare spatially resolved. As used herein, the term “spatially resolved”refers to an arrangement of two or more polypeptides on a substratewherein chemical or physical events occurring at one polypeptide can bedistinguished from those occurring at the second polypeptide. Forexample, two polypeptides affixed on a substrate are spatially resolvedif a signal from a detectable label bound to one of the polypeptides canbe unambiguously assigned to one of the polypeptides at a specificlocation on the substrate.

In one embodiment, peptides to be sequenced are affixed to a substrate.In some embodiments, the substrate is made of a material such as glass,quartz, silica, plastics, metals, hydrogels, composites, or combinationsthereof. In one embodiment, the substrate is a flat planar surface. Inanother embodiment, the substrate is 3-dimensional. In some embodiments,the substrate is a chemically derivatized glass slide or silica wafer.

In one embodiment, the substrate is made from material that does notsubstantially affect the sequencing reagents and assays describedherein. In one embodiment, the substrate is resistant to the basic andacidic pH, chemicals and buffers used for Edman degradation. Thesubstrate may also be covered with a coating. In some embodiments, thecoating is resistant to the chemical reactions and conditions used inEdman degradation. In some embodiments, the coating provides attachmentpoints for affixing polypeptides to the substrate, and/or repellingnon-specific probe adsorption. In some embodiments, the coating providesattachment points for tethering the ClickP-peptide complex.

In some embodiments, the surface of the substrate is resistant to thenon-specific adhering of polypeptides or debris, so as to minimizebackground signals when detecting the probes.

In one embodiment, the substrate made of a material that is opticallytransparent. As used herein, “optically transparent” refers to amaterial that allows light to pass through the material. In oneembodiment, the substrate is minimally- or non-autofluorescent.

In one embodiment, the peptides are affixed to the substrate. In oneembodiment, the peptides are affixed to the substrate such that theN-terminal or C-terminal end of the peptide is free to allow the bindingof the ClickP compound. Accordingly, in some embodiments the peptide isaffixed to the substrate through the N-terminal or C-terminal end of thepeptide, the N-terminal amine or the C-terminal carboxylic acid group ofthe peptide. In some embodiments, the substrate contains one or moreattachment points that permit a peptide to be affixed to the substrate.

In one embodiment, the peptides are affixed to the substrate such thatthe C-terminal end of the peptide is free to allow the binding of theClickP compound. Accordingly, in some embodiments the peptide is affixedto the substrate through the N-terminal end of the peptide, theN-terminal amine group or a side chain function group of the peptide. Insome embodiments, the substrate contains one or more attachment pointsthat permit a polypeptide to be affixed to the substrate.

In some embodiments, the peptide is affixed through a covalent bond tothe surface. For example, the surface of the substrate may contain apolyethylene glycol (PEG) or carbohydrate-based coating and the peptidesare affixed to the surface via an N-hydroxysuccinimide (NHS) ester PEGlinker.

A number of different chemistries for attaching linkers and peptides toa substrate are known in the art, for example by the use of specializedcoatings that include aldehydesilane, epoxysilane or other controlledreactive moieties. In one embodiment, the substrate is glass coated withSilane or related reagent and the polypeptide is affixed to thesubstrate through a Schiff s base linkage through an exposed lysineresidue.

In some embodiments the peptide is affixed non-covalently to thesubstrate. For example, in one embodiment the C-terminal end of thepeptide is conjugated with biotin and the substrate comprises avidin orrelated molecules. In another embodiment, the C-terminal end of apeptide is conjugated to an antigen that binds to an antibody on thesurface of the substrate. In another example, the N-terminal end of thepeptide is conjugated with biotin and the substrate comprises avidin orrelated molecules. In another embodiment, the N-terminal end of apeptide is conjugated to an antigen that binds to an antibody on thesurface of the substrate.

Additional coupling agents suitable for affixing a polypeptide to asubstrate have been described in the art (See for example, Athena L. Guoand X. Y. Zhu. The Critical Role of Surface Chemistry In ProteinMicroarrays in Functional Protein Microarrays in Drug Discovery).

In one embodiment, there are provided ClickP-amino acid complex bindersthat preferentially bind to a specific ClickP-amino acid complex or asubgroup of ClickP-amino acid complexes. As used herein the phrase“preferentially binds to a specific ClickP-amino acid complex or asubgroup of ClickP-amino acid complexes” refers to a binder with agreater affinity for a specific or subgroup of ClickP-amino acidcomplexes compared to other specific or subgroup ClickP-amino acidcomplexes. A ClickP-amino acid complex binder preferentially binds atarget ClickP-amino acid complex or a subgroup of ClickP amino acidcomplexes if there is a detectable relative increase in the binding ofthe binder to a specific or subgroup of ClickP-amino acid complexes.

In one embodiment, binders that preferentially bind to a specificClickP-amino acid complex or a subgroup of ClickP-amino acid complexesare used to identify the N-terminal amino acid of a peptide. In oneembodiment, binders that preferentially bind to a specific ClickP-aminoacid complex or a subgroup of ClickP-amino acid complexes are used tosequence a peptide. In some embodiments, the binders are detectable withsingle molecule sensitivity.

In one embodiment, binders that preferentially bind to a specificClickP-amino acid complex or a subgroup of ClickP-amino acid complexesare used to identify the C-terminal amino acid of a peptide. In oneembodiment, binders that preferentially bind to a specific ClickP-aminoacid complex or a subgroup of ClickP-amino acid complexes are used tosequence a peptide. In some embodiments, the binders are detectable withsingle molecule sensitivity.

In one embodiment, there are provided binders that selectively bind to aClickP-amino acid complex or a ClickP-amino acid derivative complex. Asused herein the phrase “selectively binds to a specific ClickP-aminoacid complex” refers to a binder with a greater affinity for a specificClickP-amino acid complex compared to other ClickP-amino acid complexes.A ClickP-amino acid complex binder selectively binds a targetClickP-amino acid complex if there is a detectable relative increase inthe binding of the binder to a specific ClickP-amino acid complex.

In one embodiment, binders that selectively bind to a ClickP-amino acidcomplex or a ClickP-amino acid derivative complex are used to identifythe N-terminal amino acid of a peptide. In one embodiment, binders thatselectively bind to a ClickP-amino acid complex or a ClickP-amino acidderivative complex are used to sequence a polypeptide. In someembodiments, the binders are detectable with single moleculesensitivity.

In one embodiment, binders that selectively bind to a ClickP-amino acidcomplex or a ClickP-amino acid derivative complex are used to identifythe C-terminal amino acid of a peptide. In one embodiment, binders thatselectively bind to a ClickP-amino acid complex or a ClickP-amino acidderivative complex are used to sequence a peptide. In some embodiments,the binders are detectable with single molecule sensitivity.

The ClickP-amino acid binders that target and recognize a specificClickP-amino acid complex or subgroup of ClickP-amino acid complexes canbe a protein or peptide, a nucleic acid a chemical or combination. Thebinders may also include components containing non-canonical amino acidand synthetic nucleotides. In one embodiment, a protein binder can be,but not limited to, an antibody, or an enzyme such as peptidases,proteases, aminoacyl tRNA synthetase, peptides or transport proteinslike lipocalin. In one embodiment, the antibody is a polyclonalantibody. In one embodiment, the antibody is a monoclonal antibody. Inone embodiment, a nucleic acid binder can be, but not limited to, anaptamer DNA, RNA or a mix of synthetic nucleotides. Aptamers are DNA/RNAwith binding properties. In one embodiment, a chemical binder can be,but not limited to amino acid reactive chemistries such as maleimide andNHS ester, heterofunctional chemicals with 2 or more differentfunctional groups, or non-covalently binding supramolecular chemistries.

In one embodiment, the plurality of binders may include 20 binders thateach selectively bind to one of the 20 natural proteinogenic aminoacids. In another embodiment, the binders include 20 binders that eachselectively bind to a derivative of one of the 20 natural proteinogenicamino acids complexed with ClickP. In one embodiment, the derivativesare phenylthiocarbamyl derivatives. In a further embodiment, the bindersinclude binders that selectively bind to post-translationally-modifiedamino acids or their derivatives complexed with ClickP. In oneembodiment, the binders include binders that selectively bind tosynthetic amino acids or their derivatives complexed with ClickP.

Detecting the binders bound to the ClickP-amino acid complex can beaccomplished by any detection method know by one of skill in the art.

In one embodiment, the binders include detectable labels. Detectablelabels suitable for use with the present invention include, but are notlimited to, labels that can be detected as a single molecule.

In one embodiment, the binders are detected by contacting the binderswith a binder-specific antibody and the binder-specific antibody is thendetected.

In some embodiments, the binders or labels are detected using magneticor electrical impulses or signals.

In some embodiments, the labels on binders are oligonucleotides.Oligonucleotide labels are read out via any method known by one of skillin the art.

In one embodiment, the binders are detected by biological or syntheticnanopores via electrical impulses or signals.

In one embodiment, the labels are optically detectable, such as labelscomprising a fluorescent moiety. Examples of optically detectable labelsinclude, but are not limited to fluorescent dyes including polystyreneshells encompassing core dyes such as FluoSpheres™ Nile Red,fluorescein, rhodamine, derivatized rhodamine dyes, such as TAN/IRA,phosphor, polymethadine dye, fluorescent phosphoramidite, TEXAS RED,green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY,120 ALEXA or a derivative or modification of any of the foregoing.Additional detectable labels include color-coded nanoparticles, orquantum dots or FluoSpheres™. In one embodiment, the detectable label isresistant to photobleaching while producing lots of signal (such asphotons) at a unique and easily detectable wavelength, with highsignal-to-noise ratio.

One or more detectable labels can be conjugated to the binder reagentsdescribed herein using techniques known to a person of skill in the art.In one embodiment, a specific detectable label (or combination oflabels) is conjugated to a corresponding binding reagent therebyallowing the identification of the binding reagent by means of detectingthe label(s). For example, one or more detectable labels can beconjugated to the binding reagents described herein either directly orindirectly.

Binders bound to a ClickP-amino acid complex affixed to the substrateare detected, thereby identifying the terminal amino acid of thepolypeptide or protein. In one embodiment, the binder is identified bydetecting a detectable label (or combination of labels) conjugated tothe binder. Methods suitable for detecting the binders described hereintherefore depend on the nature of the detectable label(s) used in themethod.

In one embodiment, the binders or labels are repeatedly detected at thatlocation using a high resolution rastering laser/scanner across apre-determined grid, unique position or path on a substrate. Thesemethods are useful for the accurate and repeated detection of signals atthe same coordinates during each sequencing cycle of the methodsdescribed herein. In some embodiments, the polypeptides are randomlyaffixed to the substrate and the detection of probes proceeds byrepeatedly scanning the substrate to identify the co-ordinates andidentities of probes bound to polypeptides affixed to the substrate.

In one embodiment, detecting the binders includes ultrasensitivedetection systems that are able to repeatedly detect signals fromprecisely the same co-ordinates on a substrate, thereby assigning thedetected sequence information to a unique polypeptide molecule affixedat that coordinate.

In one embodiment, the binders are detected using an optical detectionsystem. Optical detection systems include a charge-coupled device (CCD),near-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, light scattering, dark field microscopy,photoconversion, single and/or multiphoton excitation, spectralwavelength discrimination, fluorophore identification, evanescent waveillumination, total internal reflection fluorescence (TIRF) microscopy,super-resolution fluorescence microscopy, and single-moleculelocalization microscopy. In general, methods involve detection oflaser-activated fluorescence using a microscope equipped with a camera,sometimes referred to as high-efficiency photon detection system.Suitable photon detection systems include, but are not limited to,photodiodes and intensified CCD cameras.

In one embodiment, examples of techniques suitable for single moleculedetection of fluorescent probes include confocal laser (scanning)microscopy, wide-field microscopy, near-field microscopy, fluorescencelifetime imaging microscopy, fluorescence correlation spectroscopy,fluorescence intensity distribution analysis, measuring brightnesschanges induced by quenching/dequenching of fluorescence, orfluorescence energy transfer.

In one embodiment, the ClickP complex is cleaved from the peptide. Inone embodiment, cleaving exposes the terminus of an adjacent amino acidon the peptide, whereby the adjacent amino acid is available forreaction with a ClickP compound. Optionally, the peptide is sequentiallycleaved until the last amino acid in the peptide.

In some embodiments, the C-terminal amino acid is covalently affixed tothe substrate and is not cleaved from the substrate. In one embodiment,cleaving exposes the N-terminus of an adjacent amino acid on thepeptide, whereby the adjacent amino acid is available for reaction witha ClickP compound. Optionally, the peptide is sequentially cleaved untilthe last amino acid in the peptide (C-terminal amino acid).

In some embodiments, the N-terminal amino acid is covalently affixed tothe substrate and is not cleaved from the substrate. In one embodiment,cleaving exposes the C-terminus of an adjacent amino acid on thepeptide, whereby the adjacent amino acid is available for reaction witha ClickP compound. Optionally, the peptide is sequentially cleaved untilthe last amino acid in the peptide (N-terminal amino acid).

In one embodiment, sequential terminal degradation is used to cleave theN-terminal amino acid of the peptide. In one embodiment, sequentialterminal degradation is used to cleave the C-terminal amino acid of thepeptide. Degradation generally comprises two steps, a coupling step anda cleaving step. These steps may be iteratively repeated, each timeremoving the exposed terminal amino acid residue of a peptide.

In one embodiment terminal degradation proceeds by way of contacting thepeptide with a suitable reagent such as PITC or a PITC analogue at anelevated pH to form a N-terminal phenylthiocarbamyl derivative. Reducingthe pH, such by the addition of trifluoroacetic acid results in thecleaving the N-terminal amino acid phenylthiocarbamyl derivative fromthe polypeptide to form a free anilinothiozolinone (ATZ) derivative.This ATZ derivative may be detected. In one embodiment, ATZ derivativescan be converted to phenylthiohydantoin (PTH) derivatives by exposure toacid. This PTH derivative may be detected. In one embodiment, ATZderivatives and PTH derivatives can be converted to phenylthiocarbamyl(PTC) derivatives by exposure to a reducing agent. This PTC derivativemay be detected. In one embodiment the pH of the substrate's environmentin controlled in order to control the reactions governing the couplingand cleaving steps.

In embodiments, terminal degradation proceeds by way of contacting thepeptide with a suitable reagent such as ammonium thiocyanate afteractivation with acetic anhydride to form a C-terminalpeptidylthiohydantion derivative. Reducing the pH, with a Lewis Acidresults in the cleaving the C-terminal amino acid peptidylthiohydantionderivative by resulting in an alkylated thiohydantoin (ATH) leavinggroup from the polypeptide to form a free thiohydantion derivative. ThisATH derivative may be detected. In one embodiment, ATH derivatives canbe converted to thiohydantoin derivatives by exposure to acid. Thisthiohydantoin derivative may be detected. In one embodiment, the pH ofthe substrate's environment in controlled in order to control thereactions governing the coupling and cleaving steps.

In one embodiment, the steps of contacting the peptide with a ClickPcompound, wherein the ClickP compound binds to an N-terminal amino acidor N-terminal amino acid derivative to form a ClickP-peptide complex,tethering the ClickP-peptide complex to a substrate; cleaving theClickP-peptide complex from the peptide resulting in a ClickP-amino acidcomplex; detecting and/or identifying the amino acid of the ClickP-aminoacid complex, and releasing the ClickP-amino acid complex from thesubstrate are repeated in order to sequence the peptide. Optionally, thesteps are repeated at least 2, 5, 10, 20, 30, 50, or greater than 50times in order to sequence part of or the complete peptide. Optionallyat least: 2, 5, 10, 20 30 or 50 contiguous or discontiguous amino acidresidues of the amino acid sequence of the peptide or the full aminoacid sequence of the peptide are determined.

In one embodiment, the steps of contacting the peptide with a ClickPcompound, wherein the ClickP compound binds to an C-terminal amino acidor C-terminal amino acid derivative to form a ClickP-peptide complex,tethering the ClickP-peptide complex to a substrate; cleaving theClickP-peptide complex from the peptide resulting in a ClickP-amino acidcomplex; detecting and/or identifying the amino acid of the ClickP-aminoacid complex, and releasing the ClickP-amino acid complex from thesubstrate are repeated in order to sequence the peptide. Optionally, thesteps are repeated at least 2, 5, 10, 20, 30, 50, or greater than 50times in order to sequence part of or the complete peptide. Optionallyat least: 2, 5, 10, 20 30 or 50 contiguous or discontiguous amino acidresidues of the amino acid sequence of the peptide or the full aminoacid sequence of the peptide are determined.

In one embodiment, the method further includes washing or rinsing thesubstrate before or after any one of the steps of affixing thesubstrate, contacting the peptide with a ClickP compound, tethering theClickP-peptide complex to a substrate; cleaving the ClickP-peptidecomplex from the peptide; detecting and/or identifying the amino acid ofthe ClickP-amino acid complex; and releasing the ClickP-amino acidcomplex from the substrate. Washing or rinsing the substrate removeswaste products such as cleaved N-terminal amino acids or C-terminalamino acids, debris or previously unused reagents from the substratethat could interfere with the next step in the sequencing assay.

The methods described herein allow for the sequencing of very largenumber of peptide molecules on a single substrate or on a series ofsubstrates. Accordingly, one aspect of the invention provides forsimultaneously sequencing a plurality of affixed peptides initiallypresent in a sample. In one embodiment, the sample comprises a cellextract or tissue extract. In some embodiments, the methods describedherein may be used to analyze the peptides contained in a single cell.In a further embodiment, the sample may comprise a biological fluid suchas blood, urine or mucous. Soil, water or other environmental samplesbearing mixed organism communities are also suitable for analysis.

In one embodiment, the sample comprises a mixture of syntheticallysynthesized peptides.

In one embodiment of the description, the method includes comparing thesequence of each peptide to a reference protein sequence database. Insome embodiments, small fragments comprising 10-20 or fewer sequencedamino acid residues may be useful for detecting the identity of apeptide in a sample.

In one embodiment, the method includes de novo sequencing of peptides inorder to generate sequence information about the peptide. In anotherembodiment, the method includes determining a partial sequence or anamino acid pattern and then matching the partial sequence or amino acidpatterns with reference sequences or patterns contained in a sequencedatabase.

In one embodiment, the method includes using the sequence data generatedby the method as a molecular fingerprint or in other bioinformaticprocedures to identify characteristics of the sample, such as cell type,tissue type or organismal identity.

In addition, as each peptide affixed to the substrate is optionallymonitored individually, the method is useful for the quantitativeanalysis of protein expression. For example, in some embodiments, themethod comprises comparing the sequences of each peptide, groupingsimilar peptide sequences and counting the number of instances of eachsimilar peptide sequence. The methods described herein are thereforeuseful for molecular counting or for quantifying the number of peptidesin a sample or specific kinds of peptides in a sample.

In a further embodiment, cross-linked peptides are sequenced using themethods described herein. For example, a cross-linked protein may beaffixed to a substrate and two or more N-terminal amino acids are thenbound and sequenced. The overlapping signals that are detectedcorrespond to binders each binding the two or more terminal amino acidsat that location. In one embodiment, it is possible to deduce ordeconvolute the two multiplexed/mixed sequences via a computationalalgorithm and DB search.

In a further embodiment, the methods described herein are useful for theanalysis and sequencing of phosphopeptides. For example, polypeptides ina sample comprising phosphopeptides are affixed to a substrate viametal-chelate chemistry. The phosphopolypeptides are then sequencedaccording to the methods described herein, thereby providing sequenceand quantitative information on the phosphoproteome.

Additional multiplexed single molecule read-out and fluorescentamplification schemes can involve conjugating the binders with DNAbarcodes and amplification with hybridized chain reaction (HCR). HCRinvolves triggered self-assembly of DNA nanostructures containingfluorophores and provides multiplexed, isothermal, enzyme-free,molecular signal amplification with high signal-to-background. HCR andbranched DNA amplification can allow a large number of fluorophores tobe targeted with single-barcode precision.

EXAMPLES Example 1 Characterize and Validate ClickP Function

The ability to conjugate and cleave N-terminal amino acids wasdetermined using the ClickP compound as shown in FIG. 1. The clickablealkyne group on ClickP was also be tested to ensure ClickP can link withazide conjugates.

ClickP cleavage involved conjugating the PITC group to a peptide ofknown molecular weight and measuring the molecular weight before andafter cleavage using mass spectrometry. The expected reduction inmolecular weight by loss of one amino acid would signify a successfulcleavage. Mass spectrometry was performed on the peptide only withoutClickP, peptide with ClickP conjugated to the N-terminal amino acid, andthe cleaved peptide after ClickP removes the N-terminal amino acid.Based on the mass spectrometry results, efficiencies of the conjugationand cleavage to the N-terminal amino acid of two candidate ClickPcompounds when compared to PITC is shown in FIG. 3.

FIG. 3A compares the N-terminal conjugation efficiency of PITC, ClickP1,ClickP2 and only peptide. FIG. 3B demonstrates that ClickP1 can achievePITC N-terminal conjugation efficiency at longer reaction time periods.FIG. 3C compares the N-terminal cleavage efficiency of PITC, ClickP1,ClickP2 and only peptide. Data was collected with LCMS to determineamount reactant and the amount of product. Based on these results,ClickP candidates are capable of conjugating to the N-terminal end ofthe peptide and cleaving the N-terminal amino acid to efficienciescomparable to PITC.

The functionality of the alkyne group and its ability to conjugate toazide-substrates was determined. To demonstrate this, functionalizedbeads were either coated without peptide (control) or with immobilizedpeptides. Following coating, beads were incubated with ClickP tofacilitate conjugation to the N-terminal amino acid of the peptides.Next, the clickable group of ClickP was reacted to an azide connected toa fluorophore tetramethylrhodamine (TMR) followed by a washing step.Since the reactive azide-fluorophore should only react to the alkynegroup of ClickP, ClickP conjugated to the peptide containing beadsshould be higher in fluorescent intensity when the azide-fluorophore isintroduced. The increase in fluorescence when compared to the controlwould be indicative of a functional azide-alkyne click chemistry on theClickP reagent. The results are shown in FIG. 4.

The tethering functional group of ClickP will also be tested to ensurethat it forms a bond with functionalized surfaces for immobilizingClickP, whether it is stable under Edman conditions of high heat and lowpH, and if the reducing agent cleaves the bond. For example, the modularthiol-azide linker will be tested to ensure that it forms a disulfidebond with thiol-functionalized surfaces for conditionally immobilizingClickP. This validation will involve using the linker to form disulfidebonds on thiol-functionalized beads and using the azide group on thelinker to conjugate to an alkyne-fluorophore conjugate. Adding thereducing agent TCEP is expected to cleave the disulfide bond and releasethe linker conjugated to the fluorophore, thus reducing the fluorescentintensity. Controls would test whether disulfide bonds under Edmanconditions would cleave and release the fluorophore and also whether thefluorophore itself is stable when exposed to TCEP and Edman conditions.The fluorophore would be directly conjugated to beads and expected tomaintain the same fluorescent intensity when under high heat, low pH andexposed to the reducing agent TCEP.

Example 2: Reagent for Amino Acid Recognition (“Binder” of theClickP-Amino Acid Complex)

Single-molecule peptide or protein sequence inherently involveselucidating the amino acid composition and order. All amino acids areorganic small molecule compounds that contain amine (—NH2) and carboxyl(—COOH) functional groups, differentiated by their respective side chain(R group). The ability to identify all 20 amino acid requires a set ofreagents or methods capable of discriminating their molecular structurewith high specificity.

ClickP-based amino acid isolation solves the “local environment”problem, which is define as the interference of a binder's ability tobind to a specific terminal amino acid due to the variability ofadjacent amino acids. FIG. 6A demonstrates that the binding efficiencyof an antibody targeting tryptophan at the N-terminal amino acid isperturbed by adjacent amino acids. Binding amount was quantified bybiolayer interferometry. By removing the local environment problem withClickP, binders are intended to target ClickP-amino acids instead of theterminal amino acid. FIG. 5 shows a portion of a possible ClickPcompound bound to all 20 amino acids. In FIG. 6B, a tryptophan antibodycan discriminate ClickP-tryptophan from other amino acids. Thisindicates that binders are capable of targeting specific ClickP aminoacids.

To obtain more selective binders, portions of the ClickP-amino acidcomplexes can be used as small molecules for the development ofantibodies with high affinity and specificity.

In one method, the ClickP-amino acid complexes can be injected intorabbits to elicit an immune response against the compounds and, thereby,the production of antibodies to bind the ClickP-amino acid complexes.

Downstream, the monoclonal antibodies generated via rabbit hybridomatechnology will be tested for affinity, specificity andcross-reactivity. The antibodies secreted by the different clones willbe assayed for cross-reactivity using enzyme-linked immunosorbent assay(ELISA)29 and affinity will be measured using the label-free methodBioLayer Interferometry (BLI) 30 for measuring the kinetics ofprotein-ligand interactions.

If antibodies do not display robust affinity or specificity towardsClickP bound amino acids, directed evolution approaches can be used forimproving antibody affinity and specificity. Antibody binders can beengineered to target each amino acid isolated with ClickP using yeastdisplay, a protein engineering technique that uses the expression ofrecombinant proteins incorporated into the cell wall of yeast to screenand evolve high affinity ligands. Yeast display has been used tosuccessfully engineer antibodies that target small molecules with highaffinity. The clones generated from the rabbit hybridoma can be used toconstruct an antibody library in yeast. The library will already have abias towards the ClickP target so directed evolution via mutagenesis canintroduce novel antibody variants with improved characteristics. YeastDisplay is also capable of negative selection which helps removeantibodies that cross-react with other targets. Negative selection wouldinvolve incubating yeast expressing the antibody library with magneticbeads conjugated to non-target antigens and pulling them out ofsolution. For example, when targeting ClickP bound to one particularamino acid, the other 19 amino acids can be negatively selected againstto improve the odds of a highly specific binder.

In parallel, other binders such as enzymes or nucleic acid aptamers canbe explored in case hybridoma technology does not generate anyantibodies that target ClickP-bound amino acids. There exists 20aminoacyl-tRNA synthetase enzymes that recognize their respective aminoacids. Aminoacyl-tRNA synthetases or any other amino acid bindingprotein in nature can be used as scaffold proteins on yeast display andundergo directed evolution to select for specificity and affinitytowards respective ClickP-bound amino acids. DNA/RNA aptamers aresingle-stranded oligonucleotides capable of binding various moleculeswith high specificity and affinity. It is established that RNA is ableto form specific binding sites for free amino acids and that RNAaptamers have been evolved to change its binding specificity throughrepeated rounds of in vitro selection-amplification techniques of randomRNA pools.

Antibody binders can simply have conjugated fluorophores or secondaryantibodies conjugated to fluorophores that bind to the primary antibody,amplifying fluorescent intensity.

After binders are generated for targeting ClickP-bound amino acids, thesequencing scheme and imaging platform will be implemented on peptides,proteins and cell lysates.

Example 3: Imaging and Scaling to Proteome

Amino acids can be identified by integrating all components of ClickPisolation of N-terminal amino acids, labeling with ClickP-amino acidspecific binders, imaging, and release of ClickP for subsequent cyclesof amino acid identification. Sufficient cycles of amino acididentification will provide protein sequencing information.

Peptides will first be immobilized by the C-terminus with carboxycrosslinking chemistry. Next, ClickP binds to the N-terminal amino acidof the peptide and tethers to a functionalized substrate with theaddition of a removable group. Following N-terminal cleavage, theisolated ClickP-bound amino acid is labeled with binders, imaged andremoved.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for identifying the terminal amino acid of at least onepeptide comprising: (a) contacting the peptide with a ClickP compound,wherein the ClickP compound binds to a terminal amino acid or a terminalamino acid derivative of the peptide to form a ClickP-peptide complex,(b) tethering the ClickP-peptide complex to a substrate; (c) cleavingthe complex from the peptide thereby providing a ClickP-amino acidcomplex; and (d) detecting the ClickP-amino acid complex.
 2. The methodaccording to claim 1, wherein detecting the ClickP-amino acid complexcomprises contacting the ClickP-amino acid complex with one or moreClickP-amino acid complex binders, wherein the ClickP-amino acid complexbinder binds to a ClickP-amino acid complex or a subgroup ofClickP-amino acid complexes.
 3. The method according to claim 1, whereindetecting the ClickP-amino acid complex comprises direct detectionthrough wavelengths of light.
 4. The method according to claim 1,further comprising identifying the amino acid of the ClickP-amino acidcomplex.
 5. The method according to claim 1, wherein the ClickP compoundbinds to an N-terminal amino acid or N-terminal amino acid derivative ofthe peptide to form a ClickP-amino acid complex.
 6. The method accordingto claim 1, wherein the ClickP compound binds to a C-terminal amino acidor C-terminal amino acid derivative of the peptide to form aClickP-amino acid complex.
 7. The method according to claim 1, furthercomprising the step (e) releasing the ClickP-amino acid complex from thesubstrate.
 8. The method according to claim 7, wherein steps (a) through(e) are repeated.
 9. The method according to claim 1, wherein prior tostep (b) or (c) excess and/or unbound ClickP compound is washed away.10. The method according to claim 1, wherein the peptide is affixed tothe substrate.
 11. The method according to claim 10, wherein the peptideis affixed to the substrate through the C′-terminal carboxyl group or aside chain functional group of the peptide.
 12. The method according toclaim 10, wherein the peptide is affixed to the substrate through theN′-terminal amino group or a side chain functional group of thepolypeptide.
 13. (canceled)
 14. (canceled)
 15. The method according toclaim 1, wherein the substrate comprises a functionalized surface. 16.The method according to claim 15, wherein the functionalized surface isselected from the group consisting of an azide functionalized surface, athiol functionalized surface, alkyne, DBCO, maleimide, succinimide,tetrazine, TCO, vinyl, methylcyclopropene, a primary amine surface, acarboxylic surface, a DBCO surface, an alkyne surface, and an aldehydesurface.
 17. (canceled)
 18. (canceled)
 19. The method according to claim2, wherein the one or more ClickP-amino acid complex binders comprises:(a) one or more binders that bind to a subgroup of the 20 naturalproteinogenic amino acids complexed with ClickP; (b) one or more bindersthat bind to a subgroup of post-translationally modified amino acidscomplexed with ClickP; (c) one or more binders that bind to a derivativeof (a) or (b); or (d) combinations of binders of (a), (b) or (c). 20.The method according to claim 2, wherein the one or more ClickP-aminoacid complex binders comprises: (a) one or more binders that bind to oneof 20 natural proteinogenic amino acids complexed with ClickP; (b) oneor more binders that bind to a post-translationally modified amino acidscomplexed with ClickP; (c) one or more binders that bind to a derivativeof (a) or (b); or (d) combinations of binders of (a), (b) or (c). 21.The method according to claim 19, wherein at least one binder comprisesa detectable label.
 22. The method according to claim 20, wherein atleast one binder comprises a detectable label.
 23. The method accordingto claim 1, wherein the ClickP compound further comprises a detectablelabel.
 24. The method according to claim 1 comprising identifying theterminal amino acid of two or more peptides in a sample comprising: (a)independently affixing the two or more peptides to an attachment pointon a substrate; (b) contacting the peptides with ClickP compounds,wherein the ClickP compounds bind to a terminal amino acid or terminalamino acid derivative to form a ClickP-pepetide complex, (c) tetheringthe ClickP-peptide complexes to the substrate; (d) cleaving theClickP-peptide complexes from the peptide thereby providing aClickP-amino acid complex; and (e) detecting the ClickP-amino acidcomplexes. 25-44. (canceled)
 45. A method for sequencing of at least aportion of at least one peptide comprising: (a) contacting the peptidewith a ClickP compound, wherein the ClickP compound binds to a terminalamino acid or terminal amino acid derivative of the peptide to form aClickP-peptide complex, (b) tethering the ClickP-peptide complex to asubstrate; (c) cleaving the ClickP-peptide complex from the peptide toform a ClickP-amino acid complex; (d) detecting the ClickP-amino acidcomplex; (e) identifying the amino acid of the ClickP-amino acid complex(f) releasing the ClickP-amino acid complex from the substrate; and (g)repeating steps (a) through (f). 46-65. (canceled)
 66. The methodaccording to claim 45 comprising sequencing at least a portion of two ormore peptides in a sample comprising: (a) independently affixing the twoor more peptides to an attachment point on a substrate; (b) contactingthe two or more peptides with a ClickP compounds, wherein the ClickPcompounds bind to a terminal amino acid or terminal amino acidderivative to form a ClickP-peptide complexes, (c) tethering theClickP-peptide complexes to the substrate; (d) cleaving theClickP-peptide complexes from the peptide to form ClickP-amino acidcomplexes; (e) detecting the ClickP-amino acid complexes; (f)identifying the amino acid of the ClickP-amino acid complexes; (g)releasing the ClickP-amino acid complexes from the substrate; and (h)repeating steps (b) through (g). 67-85. (canceled)
 86. A ClickP-aminoacid complex comprising: (a) a ClickP compound bound to one of 20natural proteinogenic amino acids; (b) a ClickP compound bound to apost-translationally modified amino acid; or (c) a ClickP compound boundto a derivative of (a) or (b).
 87. A ClickP-amino acid complex bindercomprising: (a) a binder that binds to a subgroup of the 20 naturalproteinogenic amino acids complexed with ClickP; (b) a binder that bindsto a subgroup of post-translationally modified amino acids complexedwith ClickP; or (c) a binder that binds to a derivative of (a) or (b).88. A ClickP-amino acid complex binder comprising: (a) a binder thatbinds to one of 20 natural proteinogenic amino acids complexed withClickP; (b) a binder that binds to a post-translationally modified aminoacids complexed with ClickP; or (c) a binder that binds to a derivativeof (a) or (b).
 89. (canceled)